Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP5 2-K from Arabidopsis thaliana.

IF 0.9 4区 生物学
Jose Ignacio Baños-Sanz, Julia Sanz-Aparicio, Charles A Brearley, Beatriz González
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引用次数: 0

Abstract

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP(5) 2-K) is a key enzyme that catalyzes the synthesis of phytic acid (IP(6)) from inositol 1,3,4,5,6-pentakisphosphate (IP(5)) and ATP. The first structure of IP(5) 2-K, that from Arabidopsis thaliana, has been solved previously; it only crystallized in the presence of inositol, either the substrate IP(5) or the product IP(6), and failed to crystallize in its free state (without inositol). Based on structural analysis, a point mutation of IP(5) 2-K (W129A) has been produced in order to overcome this limitation and obtain information about protein conformational changes upon substrate binding. Here, the production and crystallization of W129A IP(5) 2-K in its free state and with bound nucleotide is described. These crystals differed from the native crystals and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 66.00, b = 68.23, c = 105.80 Å and a = 63.06, b = 71.80, c = 100.23 Å, respectively. The crystals diffracted to resolutions of 2.22 Å (apo) and 2.05 Å (nucleotide bound) using synchrotron radiation and contained one molecule per asymmetric unit. The structures have been determined using the molecular-replacement method and refinement is being undertaken.

拟南芥中 InsP5 2-K 的表达、纯化、结晶和初步 X 射线衍射分析。
肌醇 1,3,4,5,6-五磷酸 2-激酶(IP(5) 2-K)是催化肌醇 1,3,4,5,6-五磷酸(IP(5))和 ATP 合成植酸(IP(6))的关键酶。IP(5) 2-K 的第一个结构,即来自拟南芥的 IP(5) 2-K 结构,此前已被解决;它只有在肌醇(底物 IP(5) 或产物 IP(6))存在的情况下才能结晶,而在自由状态(不含肌醇)下则无法结晶。在结构分析的基础上,我们制作了 IP(5) 2-K 的点突变(W129A),以克服这一限制,并获得有关底物结合时蛋白质构象变化的信息。本文介绍了 W129A IP(5) 2-K 自由状态和与核苷酸结合状态的制备和结晶。这些晶体与原生晶体不同,属于正交空间群 P2(1)2(1)2,单位晶胞参数分别为 a = 66.00、b = 68.23、c = 105.80 Å 和 a = 63.06、b = 71.80、c = 100.23 Å。这些晶体在同步辐射下的衍射分辨率分别为 2.22 Å(apo)和 2.05 Å(核苷酸结合),每个不对称单元包含一个分子。这些结构是用分子置换法测定的,目前正在进行细化。
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来源期刊
自引率
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0
审稿时长
2-4 weeks
期刊介绍: Acta Crystallographica Section F is a rapid structural biology communications journal. Articles on any aspect of structural biology, including structures determined using high-throughput methods or from iterative studies such as those used in the pharmaceutical industry, are welcomed by the journal. The journal offers the option of open access, and all communications benefit from unlimited free use of colour illustrations and no page charges. Authors are encouraged to submit multimedia content for publication with their articles. Acta Cryst. F has a dedicated online tool called publBio that is designed to make the preparation and submission of articles easier for authors.
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